The objective of the proposed research is to elucidate the enzymatic mechanisms by which the extracellular matrix of human articular cartilage is degraded in osteoarthritis. All studies will concern the proteases directly present in human cartilage, not those produced in culture systems. The first aim is to purify and characterize 3 metalloprotease extracted directly from cartilage: collagenase, a neutral and an acid metalloprotease acting on proteoglycans. All 3 degrade the cartilage matrix at physiological pH, occur in latent forms, and are inhibited by TIMP, the tissue inhibitor of metalloproteases. The proteases will be characterized with respect to sensitivity to natural and synthetic inhibitors, specificity of action on natural and synthetic substrates, and relationship to known metalloproteases from other sources. Particular attention will be given to the mechanisms of activation of latent metalloproteases; we have evidence that a serine protease of the cartilage may activate metalloproteases. The second aim is to relate the levels of metalloproteases and TIMP to the development of osteoarthritis in the cartilage of tibial plateau removed at arthroplasty. Levels of proteases and inhibitors have been measured by us in the insoluble fraction of tissue homogenates. These methods will now be improved by extraction and selective assay of proteases or inhibitors on exogenous substrates and enzymes. A working hypothesis is that increased levels of proteases exceed the available TIMP in osteoarthritic tissue, resulting in matrix depletion. Immunohistochemical methods will be used to localize the proteases in cartilage and to establish their cellular origin. ELISA methods may be developed for routine quantitation of the proteases. This work on cartilage proteases and their regulation will be of considerable importance in understanding the pathogenesis of joint disease, since it will define the nature and levels of the proteases actually in the tissue, possibly arising from the chondrocytes. The knowledge of enzyme properties and specificities will be of importance in developing specific inhibitors as possible therapeutic agents. The information will also be of value in understanding cartilage remodeling, bone resorption, calcification, fracture healing, bone remodeling, osteoporosis, and related topics.